Acylated glucagon analogues

ABSTRACT

The invention provides materials and methods for promoting weight loss or preventing weight gain, and in the treatment of diabetes and associated metabolic disorders. In particular, the invention provides novel acylated glucagon analogue peptides effective in such methods. The peptides may mediate their effect by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

FIELD OF THE INVENTION

The present invention relates to acylated glucagon analogues and theirmedical use, for example in the treatment of obesity and diabetes.

BACKGROUND OF THE INVENTION

Obesity and diabetes are globally increasing health problems and areassociated with various diseases, particularly cardiovascular disease(CVD), obstructive sleep apnea, stroke, peripheral artery disease,microvascular complications and osteoarthritis.

There are 246 million people worldwide with diabetes, and by 2025 it isestimated that 380 million will have diabetes. Many have additionalcardiovascular risk factors including high/aberrant LDL andtriglycerides and low HDL.

Cardiovascular disease accounts for about 50% of the mortality in peoplewith diabetes and the morbidity and mortality rates relating to obesityand diabetes underscore the medical need for efficacious treatmentoptions.

Preproglucagon is a 158 amino acid precursor polypeptide that isdifferentially processed in the tissues to form a number of structurallyrelated proglucagon-derived peptides, including glucagon (Glu),glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), andoxyntomodulin (OXM). These molecules are involved in a wide variety ofphysiological functions, including glucose homeostasis, insulinsecretion, gastric emptying and intestinal growth, as well as regulationof food intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon and has the sequenceHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.Oxyntomodulin (OXM) is a 37 amino acid peptide which includes thecomplete 29 amino acid sequence of glucagon with an octapeptidecarboxyterminal extension (amino acids 82 to 89 of pre-proglucagon,having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala and termed“intervening peptide 1” or IP-1; the full sequence of humanoxyntomodulin is thusHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).The major biologically active fragment of GLP-1 is produced as a30-amino acid, C-terminally amidated peptide that corresponds to aminoacids 98 to 127 of pre-proglucagon.

Glucagon helps maintain the level of glucose in the blood by binding toglucagon receptors on hepatocytes, causing the liver to releaseglucose—stored in the form of glycogen—through glycogenolysis. As thesestores become depleted, glucagon stimulates the liver to synthesizeadditional glucose by gluconeogenesis. This glucose is released into thebloodstream, preventing the development of hypoglycemia. Additionally,glucagon has been demonstrated to increase lipolysis and decrease bodyweight.

GLP-1 decreases elevated blood glucose levels by improvingglucose-stimulated insulin secretion and promotes weight loss chieflythrough decreasing food intake.

Oxyntomodulin is released into the blood in response to food ingestionand in proportion to meal calorie content. The mechanism of action ofoxyntomodulin is not well understood. In particular, it is not knownwhether the effects of the hormone are mediated exclusively through theglucagon receptor and the GLP-1 receptor, or through one or more as-yetunidentified receptors.

Other peptides have been shown to bind and activate both the glucagonand the GLP-1 receptor (Hjort et al, Journal of Biological Chemistry,269, 30121-30124, 1994) and to suppress body weight gain and reduce foodintake (WO 2006/134340; WO 2007/100535; WO 2008/101017, WO 2008/152403,WO 2009/155257 and WO 2009/155258).

Stabilization of peptides has been shown to provide a betterpharmacokinetic profile for several drugs. In particular addition of oneor more polyethylene glycol (PEG) or acyl group has been shown toprolong half-life of peptides such as GLP-1 and other peptides withshort plasma stability

In WO 00/55184A1 and WO 00/55119 are disclosed methods for acylation ofa range of peptides, in particular GLP-1. Madsen et al (J. Med. Chem.2007, 50, 6126-6132) describe GLP-1 acylated at position 20(Liraglutide) and provide data on its stability.

Stabilization of OXM by PEGylation and C-terminal acylation has alsobeen shown to improve the pharmacokinetic profile of selected analoguesin WO2007/100535, WO08/071972 and in Endocrinology 2009, 150(4),1712-1721 by Druce, M R et al.

It has recently been shown that PEGylation of glucagon analogues has asignificant effect on the pharmacokinetic profile of the testedcompounds (WO2008/101017) but also interferes with the potency of thesecompounds.

SUMMARY OF THE INVENTION

The invention provides a compound having the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula I

His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-X27-X28-Ala-X30;  (I)

whereinX2 is selected from Aib or Ser;X12 is selected from Lys, Arg and Leu;X16 is selected from Arg and X;X17 is selected from Arg and X;X20 is selected from Arg, His and X;X21 is selected from Asp and Glu;X24 is selected from Ala and X;X27 is selected from Leu and X;X28 is selected from Arg and X;X30 is X or is absent;wherein at least one of X16, X17, X20, X24, X27, X28, and X30 is X;and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, Ser, Cys, Dbu, Dpr and Orn;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVEWLLRA.

X30 may be present or absent. In those embodiments when X30 is present,it may be desirable for it to be Lys.

In certain embodiments, any residue X, and especially any residue Xwhich is conjugated to a lipophilic substituent, is independentlyselected from Lys, Glu or Cys.

In certain embodiments,

X16 is selected from Glu, Lys and Ser;X17 is selected from Lys and Cys;X20 is selected from His, Lys, Arg and Cys;X24 is selected from Lys, Glu and Ala;X27 is selected from Leu and Lys; and/orX28 is selected from Ser, Arg and Lys.

Specific combinations of residues which may be present in the peptide offormula I include the following:

X2 is Aib and X17 is Lys; X2 is Aib and X17 is Cys; X2 is Aib and X20 isCys; X2 is Aib and X28 is Lys; X12 is Arg and X17 is Lys; X12 is Leu andX17 is Lys; X12 is Lys and X20 is Lys; X12 is Lys and X17 is Lys; X16 isLys and X17 is Lys; X16 is Ser and X17 is Lys; X17 is Lys and X20 isLys; X17 is Lys and X21 is Asp; X17 is Lys and X24 is Glu; X17 is Lysand X27 is Leu; X17 is Lys and X27 is Lys; X17 is Lys and X28 is Ser;X17 is Lys and X28 is Arg; X20 is Lys and X27 is Leu; X21 is Asp and X27is Leu; X2 is Aib, X12 is Lys and X16 is Ser; X12 is Lys, X17 is Lys andX16 is Ser; X12 is Arg, X17 is Lys and X16 is Glu; X16 is Glu, X17 isLys and X20 is Lys; X16 is Ser, X21 is Asp and X24 is Glu; X17 is Lys,X24 is Glu and X28 is Arg; X17 is Lys, X24 is Glu and X28 is Lys; X17 isLys, X27 is Leu and X28 is Ser; X17 is Lys, X27 is Leu and X28 is Arg;X20 is Lys, X24 is Glu and X27 is Leu; X20 is Lys, X27 is Leu and X28 isSer; X20 is Lys, X27 is Leu and X28 is Arg; X16 is Ser, X20 is His, X24is Glu and X27 is Leu; X17 is Lys, X20 is His, X24 is Glu and X28 isSer; X17 is Lys, X20 is Lys, X24 is Glu and X27 is Leu; or X17 is Cys,X20 is Lys, X24 is Glu and X27 is Leu.

It may be desirable that the peptide of formula I contains only oneamino acid of the type which is to be derivatised by addition of thelipophilic substituent. For example, the peptide may contain only oneLys residue, only one Cys residue or only one Glu residue for thelipophilic substituent to be conjugated to that residue.

The compounds of the invention may carry one or more intramolecularbridge within the peptide sequence of formula I. Each such bridge isformed between the side chains of two amino acid residues of formula Iwhich are typically separated by three amino acids in the linear aminoacid sequence (i.e. between amino acid A and amino acid A+4).

More particularly, the bridge may be formed between the side chains ofresidue pairs 16 and 20, 17 and 21, 20 and 24, or 24 and 28. The twoside chains can be linked to one another through ionic interactions, orby covalent bonds. Thus these pairs of residues may comprise oppositelycharged side chains in order to form a salt bridge by ionicinteractions. For example, one of the residues may be Glu or Asp, whilethe other may be Lys or Arg. The pairings of Lys and Glu and Lys andAsp, may also be capable of reacting to form a lactam ring.

Examples of suitable pairs of residues at positions 16 and 20 include:

X16 is Glu and X20 is Lys; X16 is Glu and X20 is Arg; X16 is Lys and X20is Glu; and X16 is Arg and X20 is Glu.

Examples of suitable pairs of residues at positions 17 and 21 include:

X17 is Arg and X21 is Glu; X17 is Lys and X21 is Glu; X17 is Arg and X21is Asp; and X17 is Lys and X21 is Asp.

Examples of suitable pairs of residues at positions 20 and 24 include:

X20 is Glu and X24 is Lys; X20 is Glu and X24 is Arg; X20 is Lys and X24is Glu; and X20 is Arg and X24 is Glu.

Examples of suitable pairs of residues at positions 24 and 28 include:

X24 is Glu and X28 is Lys; X24 is Glu and X28 is Arg; X24 is Lys and X28is Glu; and X24 is Arg and X28 is Glu.

The pairing of Lys and Glu, e.g. to form a lactam ring, may beparticularly desirable, especially between positions 24 and 28.

It will be apparent that a residue involved in an intramolecular bridgecannot also be derivatised with a lipophilic substituent. Thus, when aresidue X is involved in an intramolecular bridge, at least one of theother residues X is conjugated to a lipophilic substituent orsubstituents.

Without wishing to be bound by any particular theory, it is believedthat such intramolecular bridges stabilise the alpha helical structureof the molecule and so increase potency and/or selectivity at the GLP-1receptor and possibly also the glucagon receptor.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IIa)

whereinX12 is selected from Lys, Arg and Leu;X16 is selected from Ser and X;

X17 is X;

X20 is selected from His and X;X21 is selected from Asp and Glu;X24 is selected from Ala and Glu;X28 is selected from Ser, Lys and Arg;and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, and Cys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively, the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IIb)

whereinX12 is selected from Lys, Arg and Leu;X16 is selected from Ser and X;

X17 is X;

X20 is selected from His and X;X21 is selected from Asp and Glu;X24 is selected from Ala and Glu;X28 is selected from Ser, Lys and Arg;and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, and Cys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIIa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IIIa)

whereinX12 is selected from Lys and Arg;

X17 is X;

X20 is selected from His and X;X21 is selected from Asp and Glu;X24 is selected from Ala and Glu;X28 is selected from Ser, Lys and Arg;and wherein each residue X is independently selected from Glu, Lys, andCys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIIb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IIIb)

whereinX12 is selected from Lys or Arg;

X17 is X;

X20 is selected from His and X;X21 is selected from Asp and Glu;X24 is selected from Ala and Glu;X28 is selected from Ser, Lys and Arg;and wherein each residue X is independently selected from Glu, Lys, andCys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IVa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IVa)

whereinX12 is selected from Lys and Arg;

X17 is X;

X21 is selected from Asp and Glu;X24 is selected from Ala and Glu;X28 is selected from Ser, Lys and Arg;wherein X is selected from the group consisting of Glu, Lys, and Cys;and wherein the side chain of X is conjugated to a lipophilicsubstituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IVb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala;  (IVb)

whereinX12 is selected from Lys and Arg;

X17 is X;

X21 is selected from Asp and Glu;X24 is selected from Ala and GluX28 is selected from Ser, Lys and Arg;wherein X is selected from the group consisting of Glu, Lys, and Cys;and wherein the side chain of X is conjugated to a lipophilicsubstituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

Alternatively the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula V

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-X28;  (V)

whereinX28 is Ser or absent;

X17 is X

wherein X is selected from the group consisting of Glu, Lys, and Cys;and wherein the side chain of X is conjugated to a lipophilicsubstituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;

In certain embodiments of the invention, the peptide of formula I mayhave the sequence:

HSQGTFTSDYSKYLDSKAAHDFVEWLLRA; HSQGTFTSDYSKYLDKKAAHDFVEWLLRA;HSQGTFTSDYSKYLDSKAAKDFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWLKRA;HSQGTFTSDYSKYLDSKAAHDFVEWLLKA; HSQGTFTSDYSRYLDSKAAHDFVEWLLRA;HSQGTFTSDYSLYLDSKAAHDFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWLLRAK;HSQGTFTSDYSKYLDSKAAHDFVEWLLSAK HSQGTFTSDYSKYLDSKAAHDFVEWLKSA;HSQGTFTSDYSKYLDSKAAHDFVKWLLRA; HSQGTFTSDYSKYLDSCAAHDFVEWLLRA;HSQGTFTSDYSKYLDSCAAHDFVEWLLSA; HSQGTFTSDYSKYLDSKAACDFVEWLLRA;HSQGTFTSDYSKYLDKSAAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSAK; H-Aib-QGTFTSDYSKYLDSKAARDFVAWLLRA;H-Aib-QGTFTSDYSKYLDSKAAKDFVAWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA;H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLKA H-Aib-QGTFTSDYSKYLDSKAAKDFVAWLLSAH-Aib-QGTFTSDYSKYLDSKAAHDFVAWLLKA; H-Aib-QGTFTSDYSKYLDKKAAHDFVAWLLRA;H-Aib-QGTFTSDYSRYLDSKAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFVKWLLSA;H-Aib-QGTFTSDYSLYLDSKAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSCAAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDSKAACDFVEWLLRA;H-Aib-QGTFTSDYSKYLDK()KAAE()DFVEWLLRA;H-Aib-QGTFTSDYSKYLDSKAAHDFVE()WLLK()AH-Aib-QGTFTSDYSKYLDSKAAK()DFVE()WLLRA;H-Aib-QGTFTSDYSKYLDSK()AAHE()FVEWLLKA; orH-Aib-QGTFTSDYSKYLDSK()AAKE()FVEWLLRA.

In certain embodiments these peptides may carry a lipohilic substituentat the position marked “*” as follows:

HSQGTFTSDYSKYLDS-K*-AAHDFVEWLLRA; HSQGTFTSDYSKYLD-K*-KAAHDFVEWLLRA;HSQGTFTSDYSKYLDSKAA-K*-DFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWL-K*-RA;HSQGTFTSDYSKYLDSKAAHDFVEWLL-K*-A; HSQGTFTSDYSRYLDS-K*-AAHDFVEWLLRA;HSQGTFTSDYSLYLDS-K*-AAHDFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-K*;HSQGTFTSDYSKYLDSKAAHDFVEWLLSA-K*; HSQGTFTSDYSKYLDSKAAHDFVEWL-K*-SA;HSQGTFTSDYSKYLDSKAAHDFV-K*-WLLRA; HSQGTFTSDYSKYLDS-C*-AAHDFVEWLLRA;HSQGTFTSDYSKYLDS-C*-AAHDFVEWLLSA; HSQGTFTSDYSKYLDSKAA-C*-DFVEWLLRA;HSQGTFTSDYSKYLD-K*-SAAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA-K*;H-Aib-QGTFTSDYSKYLDS-K*-AARDFVAWLLRA;H-Aib-QGTFTSDYSKYLDSKAA-K*-DFVAWLLRA;H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL-K*-A;H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLKA;H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLRA;H-Aib-QGTFTSDYSKYLDSKAA-K*-DFVAWLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFVAWLL-K*-A;H-Aib-QGTFTSDYSKYLD-K*-KAAHDFVAWLLRA;H-Aib-QGTFTSDYSRYLDS-K*-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFV-K*-WLLSA;H-Aib-QGTFTSDYSLYLDS-K*-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-C*-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDSKAA-C*-DFVEWLLRA;H-Aib-QGTFTSDYSKYLD-S*-KAAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDK()K*AAE()DFVEWLLRA;H-Aib-QGTFTSDYSKYLDSK*AAHDFVE()WLLK()A;H-Aib-QGTFTSDYSKYLDSK*AAK()DFVE()WLLRA;H-Aib-QGTFTSDYSKYLDSK()AAHE()FVEWLLK*A; orH-Aib-QGTFTSDYSKYLDSKOAAK*E()FVEWLLRA.

Residues marked “( )” participate in an intramolecular bond, such as alactam ring.

The side chain(s) of one or more of the residues X are conjugated to alipophilic substituent. For example, one side chain of a residue X maybe conjugated to a lipophilic substituent. Alternatively, two, or evenmore than two, side chains of residues X may be conjugated to alipophilic substituent.

For example, at least one of X16, X17, X20 and X28 may be conjugated toa lipophilic substituent.

In such cases, X30 may be absent. When X30 is present, it is typicallyconjugated to a lipophilic substituent.

Thus the compound may have just one lipophilic substituent, at position16, 17, 20, 24, 27, 28 or 30, preferably at position 16, 17 or 20,particularly at position 17.

Alternatively, the compound may have precisely two lipophilicsubstituents, each at one of positions 16, 17, 20, 24, 27, 28 or 30.Preferably one or both lipophilic substituents are present at one ofpositions 16, 17 or 20.

Thus, the compound may have lipophilic substituents at positions 16 and17, 16 and 20, 16 and 24, 16 and 27, 16 and 28 or 16 and 30; at 17 and20, 17 and 24, 17 and 27, 17 and 28 or 17 and 30; at 20 and 24, 20 and27, 20 and 28 or 20 and 30; at 24 and 27, 24 and 28 or 24 and 30; at 27and 28 or 27 and 30; or at 28 and 30.

In yet further embodiments, the compound may have one or more furtherlipophilic substituents (giving three or more in total) at furtherpositions selected from positions 16, 17, 20, 24, 27, 28 or 30. Howeverit may be desirable that a maximum of two positions are derivatised inthis way.

Z¹ may comprise a hydrocarbon chain having 10 to 24 C atoms, e.g. 10 to22 C atoms, e.g. 10 to 20 C atoms. It may have at least 11 C atoms,and/or 18 C atoms or fewer. For example, the hydrocarbon chain maycontain 12, 13, 14, 15, 16, 17 or 18 carbon atoms. Thus Z¹ may be adodecanoyl, 2-butyloctanoyl, tetradecanoyl, hexadecanoyl, heptadecanoyl,octadecanoyl or eicosanoyl moiety.

Independently, where present, Z² may be or comprise one or more aminoacid residues. For example, Z² may be a γ-Glu, Glu, β-Ala or ε-Lysresidue, or a 4-aminobutanoyl, 8-aminooctanoyl or8-amino-3,6-dioxaoctanoyl moiety.

Certain combinations of Z¹ and Z² are dodecanoyl-γ-Glu,hexadecanoly-γ-Glu, hexadecanoyl-Glu, hexadecanoyl-[3-aminopropanoyl],hexadecanoyl-[8-aminooctanoyl], hexadecanoyl-ε-Lys,2-butyloctanoyl-γ-Glu, octadecanoyl-γ-Glu andhexadecanoyl-[4-aminobutanoyl].

In particular embodiments, Z has the formula:

HSQGTFTSDYSKYLD-K(Hexadecanoyl-γ-Glu)-KAAHDFVEWLL RA;HSQGTFTSDYSKYLDSKAAHDFVEWL-K(Hexadecanoyl-γ-Glu)- RA;HSQGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)-DFVEWLL RA;HSQGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl-γ-Glu)- A;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVE WLLRA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AARDFVA WLLRA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVE WLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl- γ-Glu)-A;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFV E()WLLK()A;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVE WLLKA;HSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVEWLL RA;H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)-DFVA WLLRA;H-Aib-QGTFTSDYSKYLDS-K(Dodecanoyl-γ-Glu)-AAHDFVEW LLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[3- aminopropanoyl])-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl- [8-aminooctanoyl])-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-ε-Lys)-AAHDFVEW LLSA:HSQGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLLSA;HSQGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)-AAHDFVEWLL SA;HSQGTFTSDYSKYLDS-K([2-Butyloctanoyl]-γ-Glu)- AAHDFVEWLLSA;HSQGTFTSDYSKYLDS-K(Hexadecanoyl- [4-Aminobutanoyl])-AAHDFVEWLLSA;HSQGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)-AAHDFVEWLL SA;HSQGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLL SA;H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)-AAHDFVE WLLSA;H-Aib-QGTFTSDYSKYLDS-K([2-Butyloctanoyl]-γ-Glu)- AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl[4- Aminobutanoyl])-AAHDFVEWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)-AAHDF VEWLLSA; orH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLL SA.

Residues marked “( )” participate in an intramolecular bond, such as alactam ring.

In a further embodiment, Z has the formula:

H-Aib-QGTFTSDYS-K(Hexadecanoyl-isoGlu)-YLDSKAAHDFV EWLLSA;H-Aib-QGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)-KAAHDFV EWLLSA;H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-isoGlu)-DFV EWLLSA;H-Aib-QGTFTSDYSKYLDSKAAHDFV-K(Hexadecanoyl- isoGlu)-WLLSA;H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoLys)-AARDFV AWLLRA;H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAKDFV EWLLSA;H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAHDFV EWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHEFV EWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFV EWLLSA;H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFV EWLLEA.

In a further aspect, Z has the formula:

H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDF VEWLLS;H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDF VEWLL;

In still a further aspect, Z has the formula:

H-Aib-EGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA;

The invention provides a compound having the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula I

His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-X27-X28-Ala-X30;wherein

X2 is Aib or Ser;

X12 is selected from Lys, Arg or Leu;

X16 is Arg or X; X17 is Arg or X; X20 is Arg, His or X; X21 is Asp orGlu; X24 is Ala or X; X27 is Leu or X; X28 is Arg or X;

X30 is X or is absent;and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, Ser, Cys, Dbu, Dpr and Orn;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVEWLLRA.

X30 may be present or absent. In those embodiments when X30 is present,it may be desirable for it to be Lys.

In certain embodiments, any residue X, and especially any residue Xwhich is conjugated to a lipophilic substituent, is independentlyselected from Lys, Glu or Cys.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys, Arg or Leu;

X16 is Ser or X; X17 is X; X20 is His or X; X21 is Asp or Glu; X24 isAla or Glu; X28 is Ser, Lys or Arg;

and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, or Cys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively, the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys, Arg or Leu;

X16 is Ser or X; X17 is X; X20 is His or X; X21 is Asp or Glu; X24 isAla or Glu; X28 is Ser, Lys or Arg;

and wherein each residue X is independently selected from the groupconsisting of Glu, Lys, or Cys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIIa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys or Arg;

X17 is X; X20 is His or X; X21 is Asp or Glu; X24 is Ala or Glu; X28 isSer, Lys or Arg;

and wherein each residue X is independently selected from Glu, Lys, orCys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IIIb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys or Arg;

X17 is X; X20 is His or X; X21 is Asp or Glu; X24 is Ala or Glu; X28 isSer, Lys or Arg;

and wherein each residue X is independently selected from Glu, Lys, orCys;wherein the side chain of at least one residue X is conjugated to alipophilic substituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

The compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IVa

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys or Arg;

X17 is X; X21 is Asp or Glu; X24 is Ala or Glu; X28 is Ser, Lys or Arg;

wherein X is selected from the group consisting of Glu, Lys, or Cys;and wherein the side chain of X is conjugated to a lipophilicsubstituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z².

Alternatively the compound may have the formula:

R¹—Z—R²

wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;R² is OH or NH₂;and Z is a peptide having the formula IVb

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-X21-Phe-Val-X24- Trp-Leu-Leu-X28-Ala;whereinX12 is selected from Lys or Arg;

X17 is X; X21 is Asp or Glu; X24 is Ala or Glu X28 is Ser, Lys or Arg;

wherein X is selected from the group consisting of Glu, Lys, or Cys;and wherein the side chain of X is conjugated to a lipophilicsubstituent having the formula:(i) Z¹, wherein Z¹ is a lipophilic moiety conjugated directly to theside chain of X; or(ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²;with the proviso that Z is notHSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu))-AAHDFVEWLLRA.

In a further aspect, the present invention provides a compositioncomprising a compound as defined herein, or a salt or derivativethereof, in admixture with a carrier. In preferred embodiments, thecomposition is a pharmaceutically acceptable composition and the carrieris a pharmaceutically acceptable carrier. The salt may be apharmaceutically acceptable acid addition salt of the compound, e.g. anacetate or chloride salt.

The compounds described find use in preventing weight gain or promotingweight loss. By “preventing” is meant inhibiting or reducing weight gainwhen compared to the absence of treatment, and is not necessarily meantto imply complete cessation of weight gain. The peptides may cause adecrease in food intake and/or increased energy expenditure, resultingin the observed effect on body weight. Independently of their effect onbody weight, the compounds of the invention may have a beneficial effecton circulating glucose levels, glucose tolerance, and/or on circulatingcholesterol levels, being capable of lowering circulating LDL levels andincreasing HDL/LDL ratio. Thus the compounds of the invention can beused for direct or indirect therapy of any condition caused orcharacterised by excess body weight, such as the treatment and/orprevention of obesity, morbid obesity, obesity linked inflammation,obesity linked gallbladder disease, obesity induced sleep apnea. Theymay also be used for the treatment of pre-diabetes, insulin resistance,glucose intolerance, type 2 diabetes, type I diabetes, hypertension oratherogenic dyslipidaemia (or a combination of two or more of thesemetabolic risk factors), atherosclerois, arteriosclerosis, coronaryheart disease, peripheral artery disease, stroke and microvasculardisease. Their effects in these conditions may be as a result of orassociated with their effect on body weight, or may be independentthereof.

Thus the invention provides use of a compound of the invention in thetreatment of a condition as described above, in an individual in needthereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for the treatment of a condition asdescribed above.

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity,dyslipidaemia or hypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention (or the salt thereof) can be used incombination with an anti-diabetic agent including but not limited tometformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone,or insulin. In a preferred embodiment the compound or salt thereof isused in combination with insulin, DPP-IV inhibitor, sulfonylurea ormetformin, particularly sulfonylurea or metformin, for achievingadequate glycemic control. In an even more preferred embodiment thecompound or salt thereof is used in combination with a metformin, asulfonylurea, insulin or an insulin analogue for achieving adequateglycemic control. Examples of insulin analogues include but are notlimited to Lantus, Novorapid, Humalog, Novomix, Actraphane HM, Levemirand Apidra.

The compound or salt thereof can further be used in combination with ananti-obesity agent including but not limited to a glucagon-like peptidereceptor 1 agonist, peptide YY or analogue thereof, cannabinoid receptor1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, ormelanin concentrating hormone receptor 1 antagonist.

The compound or salt thereof can further be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretic, beta-blocker, or calcium channel blocker.

The compound or salt thereof can be used in combination with ananti-dyslipidemia agent including but not limited to a statin, afibrate, a niacin or a cholesterol absorbtion inhibitor.

DESCRIPTION OF THE FIGURES

FIG. 1. Pharmacokinetic profile of compound 13 after subcutaneous (s.c.)administration to mice at a dose of 100 nmol/kg.

FIG. 2. Effect of 21 days s.c. administration of compound 11 (10nmol/kg) on oral glucose tolerance in long term high fat fed C57BL/6Jmice. Data are shown as mean±SEM.

FIG. 3. Diabetic (db/db) mice were treated with vehicle or compound 7(12.7 nmol/kg) for 4 weeks and HbAlc was determined (Cobas® applicationnote: A1C-2) in whole blood samples (20 μl) collected from the treatedmice. The ΔHbA1c (%) was calculated for each mouse by subtracting itsHbA1c (%) at start of treatment from HbA1c (%) at 4 weeks. ΔHbA1c (%) ofdb/db mice treated for 4 weeks with vehicle=100%. *(P=0.03, Studentst-test).

FIG. 4. Effect of 21 days s.c. administration of compound 11 on bodyweight in long term high fat fed C57BL/6J mice. Data are shown asmean+SEM.

FIG. 5. Diet Induced Obese (DIO) mice were treated with vehicle orcompound 7 (12.7 nmol/kg) for 4 weeks and plasma prepared from thecollected blood samples. Total cholesterol was determined in each plasmasample (Cobas®; application note CHOL2). ***(P<0.0001, Students t-test).Data are shown as mean+SEM.

FIG. 6. Diet Induced Obese (DIO) mice were treated with vehicle orcompound 7 (12.7 nmol/kg) and plasma prepared from the collected bloodsamples. LDL and HDL cholesterol were determined in each plasma sample(Cobas®; application notes HDLC3 and LDL_C). ***(P<0.0001, Studentst-test). Data are shown as mean+SEM.

FIG. 7. Effect of s.c. administration of GluGLP-1 agonists on bodyweight gain in high fat fed C57BL/6J mice. Data are mean±SEM. Blackline: Vehicle (PBS), Grey line: Low dose (0.5 nmol/kg), Broken line:High dose (5 nmol/kg).

FIG. 8. Effect of acute s.c. administration of Compound 7 on oralglucose tolerance 2, 4, 6, 8, 10 and 12 h after dosing in high fat fedC57BL/6J mice. Data are expressed as mean+SEM.

FIG. 9. Effect of s.c. administration of Compound 7 and exendin-4 onfood intake/body weight in young lean C57BL/6J mice. Data are mean+SEM.*=p<0.05 versus young lean vehicle. Data are expressed as mean+SEM.

FIG. 10. Effect of s.c. administration of Compound 7 and exendin-4 oncumulative food intake/body weight in old obese C57BL/6J mice. Data aremean+SEM. *=p<0.05 versus old obese vehicle. Data are expressed asmean+SEM.

FIG. 11. Effect of s.c. administration of Vehicle, exendin-4 (10nmol/kg) and Compound 11 (10 nmol/kg) on plasma lipid concentration inold obese C57BL/6J mice. Data are mean+SEM.

FIG. 12. Mice were treated twice daily s.c. with Compound. 1 andCompound. 11 (at two doses: 0.5 and 5 nmol/kg) or vehicle for 2 weeks.On the day of sacrifice, the liver was exposed, and weighed. Compound 1significantly increased “liver weight/body weight ratio” at the highdose. Compound. 11 did not affect “liver weight/body weight ratio” atthe two doses (0.5 and 5 nmol/kg). Compound 1 is a non-acylated dualGluGLP-1 agonists and Compound. 11 is a long-acting acylated dualGluGLP-1 agonists (FIG. 12).

FIG. 13. Diabetic (db/db) mice were treated with vehicle or compound 11(12.7 nmol/kg) for 4 weeks and HbA1c was determined (Cobas® applicationnote: A1C-2) in whole blood samples (20 μl) collected from the treatedmice. The ΔHbA1c (%) was calculated for each mouse by subtracting itsHbA1c (%) at start of treatment from HbA1c (%) at 4 weeks. ΔHbA1c (%) ofdb/db mice treated for 4 weeks with vehicle=100%. *(P=0.03, Studentst-test).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the conventional one letter and threeletter codes for naturally occurring amino acids are used, as well asgenerally accepted three letter codes for other amino acids, includingAib (α-aminoisobutyric acid), Orn (omithine), Dbu (2,4 diaminobutyricacid) and Dpr (2,3-diaminopropanoic acid).

The term “native glucagon” refers to native human glucagon having thesequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.

The peptide sequence of the compound of the invention differs from thatof native glucagon at least at positions 18, 20, 24, 27, 28 and 29. Inaddition, it may differ from that of native glucagon at one or more ofpositions 12, 16 and 17.

Native glucagon has Arg at position 18. The compound of the inventionhas the small hydrophobic residue Ala at position 18 which is believedto increase potency at both glucagon and GLP-1 receptors butparticularly the GLP-1 receptor.

The residues at positions 27, 28 and 29 of native glucagon appear toprovide significant selectivity for the glucagon receptor. Thesubstitutions at these positions with respect to the native glucagonsequence, particularly the Ala at position 29, may increase potency atand/or selectivity for the GLP-1 receptor, potentially withoutsignificant reduction of potency at the glucagon receptor. Furtherexamples which may be included in the compounds of the invention includeLeu at position 27 and Arg at position 28. Furthermore, Arg at position28 may be particularly preferred when there is a Glu at position 24 withwhich it can form an intramolecular bridge, since this may increase itseffect on potency at the GLP-1 receptor.

Substitution of the naturally-occurring Met residue at position 27 (e.g.with Leu, Lys or Glu) also reduces the potential for oxidation, therebyincreasing the chemical stability of the compounds.

Substitution of the naturally-occurring Asn residue at position 28 (e.g.by Arg or Ser) also reduces the potential for deamidation in acidicsolution, thereby increasing the chemical stability of the compounds.

Potency and/or selectivity at the GLP-1 receptor, potentially withoutsignificant loss of potency at the glucagon receptor, may also beincreased by introducing residues that are likely to stabilise analpha-helical structure in the C-terminal portion of the peptide. It maybe desirable, but is not believed essential, for this helical portion ofthe molecule to have an amphipathic character. Introduction of residuessuch as Leu at position 12 and/or Ala at position 24 may assist.Additionally or alternatively charged residues may be introduced at oneor more of positions 16, 20, 24, and 28. Thus the residues of positions24 and 28 may all be charged, the residues at positions 20, 24, and 28may all be charged, or the residues at positions 16, 20, 24, and 28 mayall be charged. For example, the residue at position 20 may be His orArg, particularly His. The residue at position 24 may be Glu, Lys orArg, particularly Glu. The residue at position 28 may be Arg.Introduction of an intramolecular bridge in this portion of themolecule, as discussed above, may also contribute to stabilising thehelical character, e.g. between positions 24 and 28.

Substitution of one or both of the naturally-occurring Gln residues atpositions 20 and 24 also reduces the potential for deamidation in acidicsolution, so increasing the chemical stability of the compounds.

A substitution relative to the native glucagon sequence at position 12(i.e. of Arg or Leu) may increase potency at both receptors and/orselectivity at the GLP-1 receptor.

C-terminal truncation of the peptide does not reduce potency of bothreceptors and/or selectivity of the GLP-1 receptor. In particular,truncation of position 29 or truncation of both position 28 and 29 doesnot reduce the receptor potency to any of the two receptors.

The side chain of one or more of the residues designated X (i.e.positions 16, 17, 20, 24, 27 and 28, and/or 30 if present) is conjugatedto a lipophilic substituent. It will be appreciated that conjugation ofthe lipophilic substituent to a particular side chain may affect (e.g.reduce) certain of the benefits which the unconjugated side chain mayprovide at that position. The inventors have found that compounds of theinvention provide a balance between the benefits of acylation and thebenefits of particular substitutions relative to the native glucagonsequence.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the compound,increase bioavailability, increase solubility, decrease adverse effects,achieve chronotherapy well known to those skilled in the art, andincrease patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behaviour in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Other groups have attempted to prolong the half life of GluGLP-1 dualagonist compounds by derivatisation with PEG (WO2008/101017). Howeversuch derivatisation appears to be most effective when applied to theC-terminus of the molecule rather than in the central core of thepeptide backbone, and potency of these compounds is still decreasedcompared to the corresponding unmodified peptide.

By contrast, the compounds of the present invention retain high potencyat both the glucagon and GLP-1 receptors while having significantlyprotracted pharmacokinetic profiles compared to the correspondingunmodified peptides.

Native glucagon has Ser at position 16. Substitution with Ala, Gly orThr has been shown to reduce adenylate cyclase activation at theglucagon receptor significantly (Unson et al. Proc. Natl. Acad. Sci.1994, 91, 454-458). Hence, derivatisation with a lipophilic substituentat position 16 would not have been expected to yield compounds retainingpotency at the glucagon receptor, as is surprisingly shown by thecompounds described in this specification. In WO2008/101017 a negativelycharged residue was found to be desirable at position 16 to minimiseloss of potency.

The presence of basic amino acids at positions 17 and 18 is generallybelieved to be necessary for full glucagon receptor activation (Unson etal. J. Biol. Chem. 1998, 273, 10308-10312). The present inventors havefound that, when position 18 is alanine, substitution with a hydrophobicamino acid in position 17 can still yield a highly potent compound. Evencompounds in which the amino acid in position 17 is derivatised with alipophilic substituent retain almost full potency at both glucagon andGLP-1 receptors, as well as displaying a significantly protractedpharmacokinetic profile. This is so even when a lysine at position 17 isderivatised, converting the basic amine side chain into a neutral amidegroup.

The present inventors have also found that compounds with acylation atposition 20 are still highly active dual agonists, despite indicationsfrom other studies that substitution in position 20 should be a basicamino acid having a side chain of 4-6 atoms in length to enhance GLP-1receptor activity compared to glucagon (WO2008/101017). The compoundsdescribed herein retain both GLP-1 and glucagon receptor activity whenposition 20 is substituted with lysine and acylated.

Peptide Synthesis

The peptide component of the compounds of the invention may bemanufactured by standard synthetic methods, recombinant expressionsystems, or any other suitable method. Thus the peptides may besynthesized in a number of ways including for example, a method whichcomprises:

(a) synthesizing the peptide by means of solid phase or liquid phasemethodology either stepwise or by fragment assembling and isolation andpurification of the final peptide product;(b) expressing a nucleic acid construct that encodes the peptide in ahost cell and recovering the expression product from the host cellculture; or(c) effecting cell-free in vitro expression of a nucleic acid constructthat encodes the peptide and recovering the expression product;or any combination of methods of (a), (b), and (c) to obtain fragmentsof the peptide, subsequently ligating the fragments to obtain thepeptide, and recovering the peptide.

It may be preferred to synthesize the analogues of the invention bymeans of solid phase or liquid phase peptide synthesis. In this context,reference is given to WO 98/11125 and, amongst many others, Fields, G Bet al., 2002, “Principles and practice of solid-phase peptidesynthesis”. In: Synthetic Peptides (2nd Edition) and the examplesherein.

Lipophilic Substituent

One or more of the amino acid side chains in the compound of theinvention is conjugated to a lipophilic substituent Z¹. Without wishingto be bound by theory, it is thought that the lipophilic substituentbinds albumin in the blood stream, thus shielding the compounds of theinvention from enzymatic degradation which can enhance the half-life ofthe compounds. It may also modulate the potency of the compound, e.g.with respect to the glucagon receptor and/or the GLP-1 receptor.

In certain embodiments, only one amino acid side chain is conjugated toa lipophilic substituent. In other embodiments, two amino acid sidechains are each conjugated to a lipophilic substituent. In yet furtherembodiments, three or even more amino acid side chains are eachconjugated to a lipophilic substituent. When a compound contains two ormore lipophilic substituents, they may be the same or different.

The lipophilic substituent Z¹ may be covalently bonded to an atom in theamino acid side chain, or alternatively may be conjugated to the aminoacid side chain by a spacer Z².

The term “conjugated” is used here to describe the physical attachmentof one identifiable chemical moiety to another, and the structuralrelationship between such moieties. It should not be taken to imply anyparticular method of synthesis.

The spacer Z², when present, is used to provide a spacing between thecompound and the lipophilic moiety.

The lipophilic substituent may be attached to the amino acid side chainor to the spacer via an ester, a sulphonyl ester, a thioester, an amideor a sulphonamide. Accordingly it will be understood that preferably thelipophilic substituent includes an acyl group, a sulphonyl group, an Natom, an O atom or an S atom which forms part of the ester, sulphonylester, thioester, amide or sulphonamide. Preferably, an acyl group inthe lipophilic substituent forms part of an amide or ester with theamino acid side chain or the spacer.

The lipophilic substituent may include a hydrocarbon chain having 10 to24 C atoms, e.g. 10 to 22 C atoms, e.g. 10 to 20 C atoms. Preferably ithas at least 11 C atoms, and preferably it has 18 C atoms or fewer. Forexample, the hydrocarbon chain may contain 12, 13, 14, 15, 16, 17 or 18carbon atoms. The hydrocarbon chain may be linear or branched and may besaturated or unsaturated. From the discussion above it will beunderstood that the hydrocarbon chain is preferably substituted with amoiety which forms part of the attachment to the amino acid side chainor the spacer, for example an acyl group, a sulphonyl group, an N atom,an O atom or an S atom. Most preferably the hydrocarbon chain issubstituted with acyl, and accordingly the hydrocarbon chain may be partof an alkanoyl group, for example a dodecanoyl, 2-butyloctanoyl,tetradecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl or eicosanoylgroup.

As mentioned above, the lipophilic substituent Z¹ may be conjugated tothe amino acid side chain by a spacer Z². When present, the spacer isattached to the lipophilic substituent and to the amino acid side chain.The spacer may be attached to the lipophilic substituent and to theamino acid side chain independently by an ester, a sulphonyl ester, athioester, an amide or a sulphonamide. Accordingly, it may include twomoieties independently selected from acyl, sulphonyl, an N atom, an Oatom or an S atom. The spacer may consist of a linear C₁₋₁₀ hydrocarbonchain or more preferably a linear C₁₋₅ hydrocarbon chain. Furthermorethe spacer can be substituted with one or more substituents selectedfrom C₁₋₆ alkyl, C₁₋₆ alkyl amine, C₁₋₆ alkyl hydroxy and C₁₋₆ alkylcarboxy.

The spacer may be, for example, a residue of any naturally occurring orunnatural amino acid. For example, the spacer may be a residue of Gly,Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln,Asn, α-Glu, γ-Glu, ε-Lys, Asp, Ser, Thr, Gaba, Aib, β-Ala (i.e.3-aminopropanoyl), 4-aminobutanoyl, 5-aminopentanoyl, 6-aminohexanoyl,7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl, 10-aminodecanoyl or8-amino-3,6-dioxaoctanoyl. In certain embodiments, the spacer is aresidue of Glu, γ-Glu, ε-Lys, β-Ala (i.e. 3-aminopropanoyl),4-aminobutanoyl, 8-aminooctanoyl or 8-amino-3,6-dioxaoctanoyl. In thepresent invention, γ-Glu and isoGlu are used interchangeably.

The amino acid side chain to which the lipophilic substituent isconjugated is a side chain of a Glu, Lys, Ser, Cys, Dbu, Dpr or Ornresidue. For example it may be a side chain of a Lys, Glu or Cysresidue. Where two or more side chains carry a lipophilic substituent,they may be independently selected from these residues. Thus the aminoacid side chain includes an carboxy, hydroxyl, thiol, amide or aminegroup, for forming an ester, a sulphonyl ester, a thioester, an amide ora sulphonamide with the spacer or lipophilic substituent.

An example of a lipophilic substituent comprising a lipophilic moiety Z¹and spacer Z² is shown in the formula below:

Here, the side chain of a Lys residue from the peptide of formula I iscovalently attached to an γ-Glu spacer (Z²) via an amide linkage. Ahexadecanoyl group (Z¹) is covalently attached to the γ-Glu spacer viaan amide linkage. This combination of lipophilic moiety and spacer,conjugated to a Lys residue, may be referred to by the short-handnotation K(Hexadecanoyl-γ-Glu), e.g. when shown in formulae of specificcompounds. γ-Glu can also be referred to as isoGlu, and a hexadecanoylgroup as a palmitoyl group. Thus it will be apparent that the notation(Hexadecanoyl-γ-Glu) is equivalent to the notations (isoGlu(Palm)) or(isoGlu(Palmitoyl)) as used for example in PCT/GB2008/004121.

The skilled person will be well aware of suitable techniques forpreparing the compounds of the invention. For examples of suitablechemistry, see WO98/08871, WO00/55184, WO00/55119, Madsen et al (J. Med.Chem. 2007, 50, 6126-32), and Knudsen et al. 2000 (J. Med Chem. 43,1664-1669).

PEGylated and/or acylation have a short half-life (T½), which gives riseto burst increases of GluGLP-1 agonist concentrations. The glucagonreceptor is thus being subjected to burst exposure to the glucagonagonism once (or twice) daily throughout the treatment period.

Without being bound to any theory repeated burst exposure of GluR toglucagon agonism seems to bring havoc to the lipid and free fatty acidtrafficking between the liver and adipose tissue with the result thatfat accumulates in the liver.

Constant exposure of GluR to glucagon agonism blocks accumulation of fatin the liver

It has thus been found, that repeated treatment with glucagon or shortacting dual GluGLP-1 agonists give rise to enlarged liver due to fat andglycogen accumulation (Chan et al., 1984. Exp. Mol. Path. 40, 320-327).

Repeated treatment with long-acting acylated dual GluGLP-1 agonists donot give rise to change in liver size (enlarged or shrunken) in normalweight subjects, but normalize liver lipid content (Day et al., 2009;Nat. Chem. Biol. 5, 749-57).

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptorsmay be used as an indication of agonist activity, but in general it ispreferred to use a biological assay which measures intracellularsignalling caused by binding of the compound to the relevant receptor.For example, activation of the glucagon receptor by a glucagon agonistwill stimulate cellular cyclic AMP (cAMP) formation. Similarly,activation of the GLP-1 receptor by a GLP-1 agonist will stimulatecellular cAMP formation. Thus, production of cAMP in suitable cellsexpressing one of these two receptors can be used to monitor therelevant receptor activity. Use of a suitable pair of cell types, eachexpressing one receptor but not the other, can hence be used todetermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. The GLP-1 receptor and/or the glucagon receptor mayhave the sequence of the receptors as described in the examples. Forexample, the assays may make use the human glucagon receptor(Glucagon-R) having primary accession number GI: 4503947 (NP_000151.1)and/or the human glucagon-like peptide 1 receptor (GLP-1R) havingprimary accession number GI:166795283 (NP_002053.3). (Where sequences ofprecursor proteins are referred to, it should of course be understoodthat assays may make use of the mature protein, lacking the signalsequence).

ECs values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀ [GLP-1R]lower than the EC₅₀ [GLP-1R] of native glucagon in a particular assaymay be considered to have higher potency at the GLP-1R than glucagon.

The compounds described in this specification are typically Glu-GLP-1dual agonists, i.e. they are capable of stimulating cAMP formation atboth the glucagon receptor and the GLP-1R. The stimulation of eachreceptor can be measured in independent assays and afterwards comparedto each other.

By comparing the EC₅₀ value for the glucagon receptor (EC₅₀[Glucagon-R]) with the EC₅₀ value for the GLP-1 receptor (EC₅₀ [GLP-1R])for a given compound the relative glucagon selectivity (%) of thatcompound can be found:

Relative Glucagon-R selectivity[Compound]=(1/EC₅₀[Glucagon-R])×100%/(1/EC₅₀ [Glucagon-R]+1/EC₅₀[GLP-1R])

The relative GLP-1R selectivity can likewise be found:

Relative GLP-1R selectivity [Compound]=(1/EC₅₀ [GLP-1R])×100%/(1/EC₅₀[Glucagon-R]+1/EC₅₀ [GLP-1R])

A compound's relative selectivity allows its effect on the GLP-1 orglucagon receptor to be compared directly to its effect on the otherreceptor. For example, the higher a compound's relative GLP-1selectivity is, the more effective that compound is on the GLP-1receptor as compared to the glucagon receptor.

Using the assays described below, we have found the relative GLP-1selectivity for human glucagon to be approximately 5%.

The compounds of the invention have a higher relative GLP-1R selectivitythan human glucagon. Thus, for a particular level of glucagon-R agonistactivity, the compound will display a higher level of GLP-1R agonistactivity (i.e. greater potency at the GLP-1 receptor) than glucagon. Itwill be understood that the absolute potency of a particular compound atthe glucagon and GLP-1 receptors may be higher, lower or approximatelyequal to that of native human glucagon, as long as the appropriaterelative GLP-1R selectivity is achieved.

Nevertheless, the compounds of this invention may have a lower EC₅₀[GLP-1R] than human glucagon. The compounds may have a lower EC₅₀[GLP-1R] than glucagon while maintaining an EC₅₀ [Glucagon-R] that isless than 10-fold higher than that of human glucagon, less than 5-foldhigher than that of human glucagon, or less than 2-fold higher than thatof human glucagon.

It may be desirable that EC₅₀ of any given compound for both theGlucagon-R and GLP-1R should be less than 1 nM.

The compounds of the invention may have an EC₅₀ [Glucagon-R] that isless than two-fold that of human glucagon. The compounds may have anEC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon andhave an EC₅₀ [GLP-1R] that is less than half that of human glucagon,less than a fifth of that of human glucagon, or less than a tenth ofthat of human glucagon.

The relative GLP-1 selectivity of the compounds may be greater than 5%and less than 95%. For example, the compounds may have a relativeselectivity of 5-20%, 10-30%, 20-50%, 30-70%, or 50-80%, or of 30-50%,40-60%, 50-70% or 75-95%.

Therapeutic Uses

The compounds of the invention may provide an attractive treatmentoption for metabolic diseases including obesity and diabetes mellitus(diabetes).

Diabetes comprises a group of metabolic diseases characterized byhyperglycemia resulting from defects in insulin secretion, insulinaction, or both. Acute signs of diabetes include excessive urineproduction, resulting compensatory thirst and increased fluid intake,blurred vision, unexplained weight loss, lethargy, and changes in energymetabolism. The chronic hyperglycemia of diabetes is associated withlong-term damage, dysfunction, and failure of various organs, notablythe eyes, kidneys, nerves, heart and blood vessels. Diabetes isclassified into type 1 diabetes, type 2 diabetes and gestationaldiabetes on the basis on pathogenetic characteristics.

Type 1 diabetes accounts for 5-10% of all diabetes cases and is causedby auto-immune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabetes accounts for 90-95% of diabetes cases and is a result ofa complex set of metabolic disorders. Type 2 diabetes is the consequenceof endogenous insulin production becoming insufficient to maintainplasma glucose levels below the diagnostic thresholds.

Gestational diabetes refers to any degree of glucose intoleranceidentified during pregnancy.

Pre-diabetes includes impaired fasting glucose and impaired glucosetolerance and refers to those states that occur when blood glucoselevels are elevated but below the levels that are established for theclinical diagnosis for diabetes.

A large proportion of people with type 2 diabetes and pre-diabetes areat increased risk of morbidity and mortality due to the high prevalenceof additional metabolic risk factors including abdominal obesity(excessive fat tissue around the abdominal internal organs), atherogenicdyslipidemia (blood fat disorders including high triglycerides, low HDLcholesterol and/or high LDL cholesterol, which foster plaque buildup inartery walls), elevated blood pressure (hypertension) a prothromboticstate (e.g. high fibrinogen or plasminogen activator inhibitor-1 in theblood), and proinflammatory state (e.g., elevated C-reactive protein inthe blood).

Conversely, obesity confers an increased risk of developingpre-diabetes, type 2 diabetes as well as e.g. certain types of cancer,obstructive sleep apnea and gall-bladder disease.

Dyslipidaemia is associated with increased risk of cardiovasculardisease. High Density Lipoprotein (HDL) is of clinical importance sincean inverse correlation exists between plasma HDL concentrations and riskof atherosclerotic disease. The majority of cholesterol stored inatherosclerotic plaques originates from LDL and hence elevatedconcentrations Low Density Lipoproteins (LDL) is closely associated withatherosclerosis. The HDL/LDL ratio is a clinical risk indictor foratherosclerosis and coronary atherosclerosis in particular.

Without wishing to be bound by any particular theory, it is believedthat the compounds of the invention act as GluGLP-1 dual agonists. Thedual agonist may combine the effect of glucagon e.g. on fat metabolismwith the effect of GLP-1 e.g. on blood glucose levels and food intake.They might therefore act to accelerate elimination of excessive adiposetissue, induce sustainable weight loss, and improve glycaemic control.Dual GluGLP-1 agonists might also act to reduce cardiovascular riskfactors such as high cholesterol and LDL-cholesterol.

The compounds of the present invention can therefore be used aspharmaceutical agents for preventing weight gain, promoting weight loss,reducing excess body weight or treating obesity (e.g. by control ofappetite, feeding, food intake, calorie intake, and/or energyexpenditure), including morbid obesity, as well as associated diseasesand health conditions including but not limited to obesity linkedinflammation, obesity linked gallbladder disease and obesity inducedsleep apnea. The compounds of the invention may also be used fortreatment of insulin resistance, glucose intolerance, pre-diabetes,increased fasting glucose, type 2 diabetes, hypertension, dyslipidemia(or a combination of these metabolic risk factors), atherosclerois,arteriosclerosis, coronary heart disease, peripheral artery disease andstroke. These are all conditions which can be associated with obesity.However, the effects of the compounds of the invention on theseconditions may be mediated in whole or in part via an effect on bodyweight, or may be independent thereof.

Pharmaceutical Compositions

The compounds of the present invention, or salts thereof, may beformulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the invention, or a salt thereof, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompases any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to the salt of thecompounds. Salts include pharmaceutically acceptable salts such as acidaddition salts and basic salts. Examples of acid addition salts includehydrochloride salts, citrate salts and acetate salts. Examples of basicsalts include salts where the cation is selected from alkali metals,such as sodium and potassium, alkaline earth metals such as calcium, andammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designatesoptionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are described in“Remington's Pharmaceutical Sciences”, 17th edition. Ed. Alfonso R.Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 andmore recent editions, and in the Encyclopaedia of PharmaceuticalTechnology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. By treatment is meant inhibiting orreducing an increase in pathology or symptoms (e.g. weight gain,hyperglycaemia) when compared to the absence of treatment, and is notnecessarily meant to imply complete cessation of the relevant condition.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Pharmaceutically acceptablecarriers or diluents include those used in formulations suitable fororal, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the compounds described herein.

Combination Therapy

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity,dyslipidaemia or hypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention (or the salt thereof) can be used incombination with an anti-diabetic agent including but not limited tometformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone,or insulin. In a preferred embodiment the compound or salt thereof isused in combination with insulin, DPP-IV inhibitor, sulfonylurea ormetformin, particularly sulfonylurea or metformin, for achievingadequate glycemic control. In an even more preferred embodiment thecompound or salt thereof is used in combination with insulin or aninsulin analogue for achieving adequate glycemic control. Examples ofinsulin analogues include but are not limited to Lantus, Novorapid,Humalog, Novomix, Actraphane HM, Levemir and Apidra.

The compound or salt thereof can further be used in combination with ananti-obesity agent including but not limited to a glucagon-like peptidereceptor 1 agonist, peptide YY or analogue thereof, cannabinoid receptor1 antagonist, lipase inhibitor, melanocortin receptor 4 agonist, ormelanin concentrating hormone receptor 1 antagonist.

The compound or salt thereof can be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretics, beta-blocker, or calcium channel blocker.

The compound or salt thereof can be used in combination with ananti-dyslipidaemia agent including but not limited to a statin, afibrate, a niacin and/or a cholesterol absorbtion inhibitor.

Methods General Synthesis of Acylated Glucagon Analogues

Solid phase peptide synthesis was performed on a CEM Liberty PeptideSynthesizer using standard Fmoc chemistry. TentaGel S Ram resin (1 g;0.25 mmol/g) was swelled in NMP (10 ml) prior to use and transferredbetween tube and reaction vessel using DCM and NMP.

Coupling:

An Fmoc-amino acid in NMP/DMF/DCM (1:1:1; 0.2 M; 5 ml) was added to theresin in a CEM Discover microwave unit together with HATU/NMP (0.5 M; 2ml) and DIPEA/NMP (2.0 M; 1 ml). The coupling mixture was heated to 75°C. for 5 min while nitrogen was bubbled through the mixture. The resinwas then washed with NMP (4×10 ml).

Deprotection:

Piperidine/NMP (20%; 10 ml) was added to the resin for initialdeprotection and the mixture was heated by microwaves (30 sec.; 40° C.).The reaction vessel was drained and a second portion of piperidine/NMP(20%; 10 ml) was added and heated (75° C.; 3 min.) again. The resin wasthen washed with NMP (6×10 ml).

Side Chain Acylation:

Fmoc-Lys(ivDde)-OH or alternatively another amino acid with anorthogonal side chain protective group was introduced at the position ofthe acylation. The N-terminal of the peptide backbone was thenBoc-protected using Boc₂O or alternatively by using a Boc-protectedamino acid in the last coupling. While the peptide was still attached tothe resin, the orthogonal side chain protective group was selectivelycleaved using freshly prepared hydrazine hydrate (2-4%) in NMP for 2×15min. The unprotected lysine side chain was first coupled withFmoc-Glu-OtBu or another spacer amino acid, which was deprotected withpiperidine and acylated with a lipophilic moiety using the peptidecoupling methodology as described above.

Abbreviations employed are as follows:

-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   DCM: dichloromethane-   DMF: N,N-dimethylformamide-   DIPEA: diisopropylethylamine-   EtOH: ethanol-   Et₂O: diethyl ether-   HATU:    N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium    hexafluorophosphate N-oxide-   MeCN: acetonitrile-   NMP: N-methylpyrrolidone-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane

Cleavage:

The resin was washed with EtOH (3×10 ml) and Et₂O (3×10 ml) and dried toconstant weight at room temperature (r.t.). The crude peptide wascleaved from the resin by treatment with TFA/TIS/water (95/2.5/2.5; 40ml, 2 h; r.t.). Most of the TFA was removed at reduced pressure and thecrude peptide was precipitated and washed three times with diethyletherand dried to constant weight at room temperature.

HPLC Purification of the Crude Peptide:

The crude peptide was purified to greater than 90% by preparativereverse phase HPLC using a PerSeptive Biosystems VISION Workstationequipped with a C-18 column (5 cm; 10 μm) and a fraction collortor andrun at 35 ml/min with a gradient of buffer A (0.1% TFA, aq.) and bufferB (0.1% TFA, 90% MeCN, aq.). Fractions were analysed by analytical HPLCand MS and relevant fractions were pooled and lyophilised. The finalproduct was characterised by HPLC and MS.

Generation of Cell Lines Expressing Human Glucagon- and GLP-1 Receptors

The cDNA encoding either the human glucagon receptor (Glucagon-R)(primary accession number P47871) or the human glucagon-like peptide 1receptor (GLP-1R) (primary accession number P43220) were cloned from thecDNA clones BC104854 (MGC:132514/IMAGE:8143857) or BC112126 (MGC:138331/IMAGE:8327594), respectively. The DNA encoding the Glucagon-R orthe GLP-1R was amplified by PCR using primers encoding terminalrestriction sites for subcloning. The 5′-end primers additionallyencoded a near Kozak consensus sequence to ensure efficient translation.The fidelity of the DNA encoding the Glucagon-R and the GLP-1R wasconfirmed by DNA sequencing. The PCR products encoding the Glucagon-R orthe GLP-1R were subcloned into a mammalian expression vector containinga neomycin (G418) resistance marker.

The mammalian expression vectors encoding the Glucagon-R or the GLP-1Rwere transfected into HEK293 cells by a standard calcium phosphatetransfection method. 48 hr after transfection cells were seeded forlimited dilution cloning and selected with 1 mg/ml G418 in the culturemedium. Three weeks later 12 surviving colonies of Glucagon-R and GLP-1Rexpressing cells were picked, propagated and tested in the Glucagon-Rand GLP-1R efficacy assays as described below. One Glucagon-R expressingclone and one GLP-1R expressing clone were chosen for compoundprofiling.

Glucagon Receptor and GLP-1 Receptor Efficacy Assays

HEK293 cells expressing the human Glucagon-R, or human GLP-1R wereseeded at 40,000 cells per well in 96-well microtiter plates coated with0.01% poly-L-lysine and grown for 1 day in culture in 100 μl growthmedium. On the day of analysis, growth medium was removed and the cellswashed once with 200 μl Tyrode buffer. Cells were incubated in 100 μlTyrode buffer containing increasing concentrations of test peptides, 100μM IBMX, and 6 mM glucose for 15 min at 37° C. The reaction was stoppedby addition of 25 μl 0.5 M HCl and incubated on ice for 60 min. The cAMPcontent was estimated using the FlashPlate® cAMP kit from Perkin-Elmer.EC₅₀ and relative efficacies compared to reference compounds (glucagonand GLP-1) were estimated by computer aided curve fitting.

Bioanalytical Screening-Method for Quantification of Peptide Glu-GLP1Agonists in Mouse Plasma after Subcutaneous Administration

Mice were dosed 100 nmol/kg subcutaneously (s.c.). The mice weresacrificed and the blood collected at the following time points; 0.5, 2,4, 6, 16 and 24 h. Plasma samples were analyzed using proteinprecipitation, followed by solid phase extraction (SPE) and liquidchromatography mass spectrometry (LC-MS).

Oral Glucose Tolerance Test (OGTT), Blood Lipids and Body Weight in HighFat Fed C57Bl/6J Normal Mice and HbA1c in db/db Mice

Male mice (Long term high fat fed C57Bl/6J, short term high fat fedC57Bl/6J and db/db) were acclimatized with free access to food andwater. They were housed in groups of 5-6 in a light-, temperature-, andhumidity-controlled room (12-hour light:12-hour dark cycle, lightsOn/Off at 2000/0800 hour; 24° C.; 50% relative humidity).

The animals were injected s.c. with 100 μl vehicle (once a day) for aperiod of three days to acclimatize the animals to handling andinjections. Blood samples were taken from the eye or from the tip of thetail. The animals were randomized before treatment.

Mice were treated twice daily s.c. with GluGLP-1 agonist or vehicle(injection volume=2.5 ml/kg). Throughout the study, body weights wererecorded daily and used to administer the body weight-corrected doses ofpeptide. Peptide solutions were prepared fresh immediately beforedosing.

Oral glucose tolerance tests (OGTT) were performed after subjecting theanimals to a short fast. To prevent confounding food intake, the animalswere kept fasted during the OGTTs. After peptide dosing an initial bloodsample was taken. Thereafter an oral dose of glucose (1 g/kg), dissolvedin phosphate buffer (pH=7.4) was given (5 ml/kg), and the animals werereturned to their home cages (t=0). The whole blood glucose (BG) wasmeasured at t=15 min, t=30 min, t=60 min, t=90 min and t=120 min.

The BG concentration was analyzed by the immobilized glucose oxidasemethod using a drop of blood (<5 μl; Elite Autoanalyser, Bayer, Denmark)following the manufacturer's instructions.

HbA1c Determination

It is possible to assess the long term effect of a compound on asubject's glucose level by determining the level of haemoglobin A1C(HbA1c). HbA1c is a glycated form of haemoglobin whose level in a cellreflects the average level of glucose to which the cell has been exposedduring its lifetime. In mice, HbA1c is a relevant biomarker for theaverage blood glucose level during the preceding 4 weeks, becauseconversion to HbA1c is limited by the erythrocyte's life span ofapproximately 47 days (Abbrecht & Littell, 1972; J. Appl. Physiol. 32,443-445). The HbA1c determination is based on Turbidimetric INhibitionImmunoAssay (TINIA) in which HbA1c in the sample reacts with anti-HbA1cto form soluble antigen-antibody complexes. Additions of polyhaptensreact with excess anti-HbA1c antibodies to form an insolubleantibody-polyhapten complex, which can be measured turbidimetrically.Liberated hemoglobin in the hemolyzed sample is converted to aderivative having a characteristic absorption spectrum, which ismeasured bichromatically during the preincubation phases. The finalresult is expressed as percent HbA1c of total hemoglobin(Cobas®Application note A1C-2).

Cholesterol Level Determination

The assay is an enzymatic colorimetric method. In the presence ofmagnesium ions, dextran sulfate selectively forms water-solublecomplexes with LDL, VLDLA and chylomicrons, which are resistant toPEG-modified enzymes. The HDL cholesterol is determined enzymatically bycholesterol esterase and cholesterol oxidase coupled with PEG to theamino groups. Cholesterol esters are broken down quantitatively to freecholesterol and fatty acids. HDL cholesterol is enzymatically oxidizedto choles-4-en-3-one and H₂O₂, and the formed H₂O₂ is measuredcolorimetrically (Cobas®; Application note HDLC3).

The direct determination of LDL takes advantage of the selectivemicellary solubilization of LDL by a nonionic detergent and theinteraction of a sugar compound and lipoproteins (VLDL andchylomicrons). The combination of a sugar compound with detergentenables the selective determination of LDL in plasma. The test principleis the same as that of cholesterol and HDL, but due to the sugar anddetergent only LDL-cholesterol esters are broken down to freecholesterol and fatty acids. Free cholesterol is then oxidized and theformed H₂O₂ is measured colorimetrically (Application note LDL_C,Cobas®).

Body Weight Gain in High Fat Fed C57BL/6J Mice.

C57Bl/6J male mice, 6 weeks old, were acclimatized in their newenvironment for 4 weeks with free access to high fat diet (HFD) (D12492,Research Diet Inc., New Brunswick, USA) and water. The animals wereinjected s.c. with 100 μl vehicle for a period of three days toacclimatize the animals to handling and injections, prior to initiationof peptide treatment. The mice were treated twice daily s.c. withexendin-4, Compound 3, Compound 6, Compound 7, Compound 8, Compound 11and Compound 12 or vehicle. Throughout the study, body weights wererecorded daily and used to administer the body weight-corrected doses ofpeptide. All animals were sacrificed on the same day by cervicaldislocation.

Oral Glucose Tolerance 2, 4, 6, 8, 10 and 12 h after Dosing in High FatFed C57Bl/6J Mice

C57Bl/6J male mice, 6 weeks old, were acclimatized to their newenvironment with free access to a high fat diet (012492, Research DietInc., New Brunswick, USA) and water. The animals were injected s.c. withvehicle for a period of three days to acclimatize the animals tohandling and injections. Blood samples were taken from the tip of thetail and blood glucose measured. The blood glucose (mM) concentrationwas analyzed by the immobilized glucose oxidase method using a drop ofblood (<5 μl; Contour Autoanalyser, Bayer, Denmark) following themanufacturers manual. After 4 weeks on the high fat diet the animalswere weighed and the body weight was used to administer a bodyweight-corrected dose of peptide. An oral glucose tolerance test (OGTT)was performed after subjecting the animals to 4 hours of fasting. At 2,4, 6, 8, 10 and 12 hours after single peptide or vehicle dosing aninitial blood sample were taken (t=−0 min). Immediately thereafter, anoral dose of glucose (1 g/kg) was given and the animals were returned totheir home cages (t=0). BG levels were measured at t=15 min, t=30 min,t=60 min and t=90 min. Immediately following blood sampling, all animalswere sacrificed by CO₂ anesthesia followed by cervical dislocation.

Food Intake in Young Lean and Old Obese C57Bl/6J Mice.

C57BL/6J mice were on a high fat diet for 11 days and C57BL/6J mice wereon a high fat diet for 52 weeks.

3 days before study, the mice were transferred to individual cages andweighed. 4 days before study, they were acclimatized to handling andtreatment by dairy s.c. injections. On the day before the experimentfood was removed at 20:00. On the day of the experiment, the mice wereweighed and treated with s.c. injections of Exendin-4, Compound 7 orVehicle at t=0 h (8:00) and t=12 h (20:00). Immediately after treatment(t=0), pre-weighed food were introduced to the mice and the cumulativefood intake was measured by weighing the remaining food after t=1, 2, 4,8, 12 and 24 hours. After weighing the food and the animals at t=24 h,the mice were sacrificed by cervical dislocation.

Hepatocyt cAMP Formation.

Experimental Procedure

Primary human hepatocyts provided by Lonza Walkersvill, Inc. werecarefully washed in TB buffer and incubated at 37° C. with peptidesdissolved in TB buffer supplemented with 100 μM IBMX and 0.1% casein for15 minutes. Prior to addition to the cells, the peptide dilutions werepre-warmed to 37° C. The reaction was stopped by addition of 25 μl ofice cold 0.5 M HCl, and the cells were incubated on ice for 60 min. ThecAMP content in the wells was determined by adding 25 μl of the acidextracts from the wells to 75 μl sodium acetate buffer, pH 6.2, in96-well microtiter “FlashPlates” coated with scintillant and anti-cAMPantibodies. Following addition of 100 μl of 10 μCi [¹²⁵I]cAMP solutionto each well, the plates were incubated overnight at 4° C., emptied, andthe amount of [¹²⁵I]cAMP bound to the Flash Plates was counted using theprogram “[¹²⁵I]cAMP flashplate 10 min” on the TopCount NXT.

Peptides were tested at a concentration range of 0.1-1000 nM.

Data Analysis and Statistics

The amount of cAMP produced by the cells was calculated by extrapolationto a cAMP standard curve.

EC₅₀ values were estimated by fitting the cAMP data to the below formulausing Sigma Plot:

${{{cAMP}\mspace{14mu} {response}} = {\frac{\left( {{cAMP}_{\max} - {cAMP}_{\min}} \right) \times c}{c + {EC}_{50}} + {cAMP}_{\min}}},$

The invention is further illustrated by the following examples.

Liver Weight/Body Weight of C57BL/6J Mice.

Mice were treated twice daily s.c. with Cpd. 1 and Cpd. 11 (at twodoses: 0.5 and 5 nmol/kg) or vehicle for 2 weeks. Throughout the study,body weights were recorded daily and used to administer the bodyweight-corrected doses of peptide. On the day of sacrifice, the liverwas exposed, and weighed.

EXAMPLES Example 1: Synthesis of Compounds and Peptide PropertiesSynthesis Example

Compound 9 was synthesized on a CEM Liberty Peptide Synthesizer usingTentaGel S Ram resin (1.17 g; 0.23 mmol/g) and Fmoc-chemistry asdescribed above. Fmoc-Lys(ivDde)-OH was used in position 17 andpseudoprolines Fmoc-Phe-Thr(.Psi. Me, Me pro)-OH andFmoc-Asp(OtBu)-Ser(.Psi., Me, Me pro)-OH were used in the peptidebackbone. After completion of the peptide backbone on the resin theN-terminal Fmoc-group was cleaved manually followed by Boc-protectionusing Boc₂O (226 mg) and DIEA (54 μl) in DCM. The ivDde-group was thencleaved with freshly prepared hydrazine hydrate/NMP (4%; 2×15 min.).Back on the CEM Liberty Peptide Synthesizer the remaining two buildingblocks, Fmoc-Glu-OtBu and hexadecanoic acid, were added to theunprotected lysine side chain.

The peptide was cleaved from the resin as described above, and thepurification was performed on a Gemini-NX column (5 cm, 10 μm, C18) witha 35 ml/min flow of a mixture of buffer A (0.1% TFA, aq.) and buffer B(0.1% TFA, 90% MeCN, aq.). The product was eluted with a linear gradientfrom 25% to 65% buffer B over 47 min., and fractions (9 ml) werecollected by a fraction collector. Relevant fractions were analysed byanalytical HPLC and MS and fractions with purities above 95% were pooledand lyophilised to a white powder. The 72 mg yield had a purity of 97%determined by analytical HPLC and the mass was 3697.05 Da as determinedby MS (Calc. 3696.97 Da).

Example 2: Efficacy on GLP-1 and Glucagon Receptors

Efficacy of the GluGLP-1 agonists were estimated by exposing cellsexpressing hGlucagonR and hGLP-1R to the listed acylated compounds atincreasing concentrations and measuring the formed cAMP as described inMethods.

Results are shown in Table 1:

TABLE 1 EC₅₀ values of acylated compounds at GLP-1 and Glucagonreceptors EC₅₀ EC₅₀ (nM) (nM) Sequence Compound GLP-1R GluRH-HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-NH2 Compound 1 0.06 0.06H-HSQGTFTSDYSKYLD-K(Hexadecanoyl-γ-Glu)- Compound 2 0.20 0.13KAAHDFVEWLLRA-NH2 H-HSQGTFTSDYSKYLD-S-K(Hexadecanoyl-γ-Glu)- Compound 30.11 0.12 AAHDFVEWLLRA-NH2 H-HSQGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)-Compound 4 0.10 0.04 DFVEWLLRA-NH2H-HSQGTFTSDYSKYLDSKAAHDFVEWL-K(Hexadecanoyl-γ- Compound 5 0.57 0.22Glu)-RA-NH2 H-HSQGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl- Compound 60.09 0.10 γ-Glu)-A-NH2 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-Compound 7 0.11 0.16 AAHDFVEWLLSA-NH2H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- Compound 9 0.12 0.17AARDFVAWLLRA-NH2 H-H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)-Compound 0.15 0.63 DFVAWLLRA-NH2 10H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- Compound 0.09 0.16AAHDFVEWLLRA-NH2 11 H-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL- Compound 0.270.27 K(Hexadecanoyl-γ-Glu)-A-NH2 12H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- Compound 0.08 0.26AAHDFVE()WLLK()A-NH2 13 H-H-Aib-QGTFTSDYSKYLDS-K(Dodecanoyl-γ-Glu)-Compound 0.14 0.78 AAHDFVEWLLSA-NH2 14H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[3- Compound 0.23 1.87Aminopropanoyl])-AAHDFVEWLLSA-NH2 15H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[8- Compound 0.24 0.46Aminooctanoyl])-AAHDFVEWLLSA-NH2 16H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-ε-Lys)- Compound 0.09 0.39AAHDFVEWLLSA-NH2 17

The residues marked ( ) form an intramolecular lactam ring.

TABLE 1a EC₅₀ values of additional acylated compounds according to theinvention EC₅₀ EC₅₀ (nM) (nM) Sequence Compound GLP-1R GluRH-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- Compound 0.066 0.091AAHDFVEWLLS-OH 18 H-H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-Compound 0.048 0.483 AAHDFVEWLL-OH 19H-H-Aib-EGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- Compound 0.057 13.266AAHDFVEWLLSA-OH 20 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-Compound 0.077 0.150 AAHDFVEWLLSA-OH 21H-H-Aib-EGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- Compound 0.014 26.370AAHDFVEWLLSA-NH2 22 H-H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl)- Compound0.140 0.124 AAHDFVEWLLSA-NH2 23H-H-Alb-QGTFTSDYSKYLDS-K([2-Butyloctanoyl]-isoGlu)- Compound 0.161 0.133AAHDFVEWLLSA-NH2 24 H-H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl-isoGlu)-Compound 0.069 0.103 AAHDFVEWLLSA-NH2 25H-H-Aib-QGTFTSDYSKYLDS-K(Dodecanoyl-isoGlu)- Compound 0.097 0.116AAHDFVEWLLSA-NH2 26 H-H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-[4- Compound0.152 0.147 Aminobutanoyl])-AAHDFVEWLLSA-NH2 27H-H-Alb-QGTFTSDYSKYLDS-K(Hexadecanoyl-E)- Compound 0.149 0.108AAHDFVEWLLSA-NH2 28 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[8- Compound0.199 0.123 Aminooctanoyl])-AAHDFVEWLLSA-NH2 29H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoLys)- Compound 0.132 0.110AAHDFVEWLLSA-NH2 30 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[3- Compound0.103 0.151 Aminopropanoyl])-AAHDFVEWLLSA-NH2 31H-H-Aib-QGTFTSDYSKYLDS-Orn(Hexadecanoyl-isoGlu)- Compound 0.195 0.193AAHDFVEWLLSA-NH2 32 H-H-Aib-QGTFTSDYS-K(Hexadecanoyl-isoGlu)- Compound0.131 0.389 YLDSKAAHDFVEWLLSA-NH2 33H-H-Aib-QGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)- Compound 0.109 0.053KAAHDFVEWLLSA-NH2 34 H-H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-isoGlu)-Compound 0.202 0.180 DFVEWLLSA-NH2 35H-H-Aib-QGTFTSDYSKYLDSKAAHDFV-K(Hexadecanoyl- Compound 0.191 0.213isoGlu)-WLLSA-NH2 36 H-H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL- Compound 0.2070.147 K(Hexadecanoyl-isoGlu)-A-NH2 37H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoLys)- Compound 0.132 0.183AARDFVAWLLRA-NH2 38 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-Compound 0.16 0.24 AAKDFVEWLLSA-NH2 39H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- Compound 0.20 0.18AAHDFVEWLLSA-NH2 40 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-Compound 0.13 0.08 AAHEFVEWLLSA-NH2 41H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- Compound 0.03 0.27AAEDFVEWLLSA-NH2 42 H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-Compound 0.082 0.12 AAHDFVEWLLEA-NH2 43

For compound 28H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLLSA-NH2 could also bewritten as H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-αGlu)-AAHDFVEWLLSA-NH2

Example 3: Pharmacokinetic Screening

Pharmacokinetic profiles were determined for various acylated compounds.Calculated T_(1/2) values are shown in Table 2, compared to(non-acylated) compound 1.

TABLE 2 Compound T_(1/2) (h) 1 0.23 2 5.8 5 5.3 4 2.0* 6 4.8 7 3.4 92.4* 11 4.9 12 6.0 13 6.4 *Only two time points were used forcalculation of T½.

All of the acylated compounds have improved T½ compared to compound 1.

A sample pharmacokinetic profile, for compound 13, is shown in FIG. 1.

Example 4: Oral Glucose Tolerance Test in DIO Mice

Effect of 21 days s.c. administration of compound 11 (10 nmol/kg) onoral glucose tolerance in long term high fat-fed C57BL/6J mice. Highfat-fed mice were fasted and an initial blood sample taken to determinefasting blood glucose level (t=0). An oral dose of glucose (1 g/kg in 5ml/kg) was then given and blood glucose levels were measured at t=30min, t=60 min, t=90 min and t=120 min. Compound 11 significantlyimproved glucose tolerance (two way ANOVA). Data are shown as mean±SEM.

Example 5: HbA1c in db/db Mice after 28 Days

Diabetic (db/db) mice were treated with vehicle or compound 7 for 4weeks, and HbA1c was determined (Cobas® application note: A1C-2) inwhole blood samples (20 μl) collected from the treated mice. Results areshown in FIG. 3. The ΔHbA1c (%) was calculated for each mice bysubtracting its HbA1c (%) at start of treatment from HbA1c (%) at 4weeks. Treatment with compound 7 decreased ΔHbA1c (%) significantly.(P=0.03; Students t-test) compared to vehicle.

Example 6: Reduced Body Weight

Effect of 21 days s.c. administration of compound 11 on body weight wasdetermined in long term high fat-fed C57BL/6J mice. C57Bl/6J male miceon high fat diet (HFD) were treated (b.i.d.; s.c.) with compound 11 (10nmol/kg) or vehicle. Body weights were recorded daily and used toadminister the body weight-corrected doses of peptide throughout thestudy. Data are shown as mean±SEM in FIG. 4. Compound 11 significantlydecreased body weight (p<0.05).

Example 7: Total Cholesterol and HDL/LDL Ratio

Diet Induced Obese (DIO) mice were treated with vehicle or compound 7for 4 weeks and plasma prepared from the collected blood samples. Thetotal cholesterol, LDL and HDL were determined in each plasma sample(Cobas® application notes: CHOL2, HDLC3 and LDL_C) and results are shownin FIGS. 5 and 6. Treatment with compound 7 significantly (P<0.0001,Students t-test) decreased total cholesterol concentrations (FIG. 5) andsignificantly (P<0.0001, Students t-test) increased the HDL/LDL-ratio(FIG. 6).

Example 8: Body Weight Gain in High Fat Fed C57BL/6J Mice

Effect of 10 days s.c. administration of Exendin-4, Compound 8, Compound3, Compound 7, Compound 11, Compound 12 and Compound 6 short term highfat-fed C57BL/6J mice. C57Bl/6J male mice on high fat diet (HFD) weretreated (b.i.d.; s.c.) (0.5 and 5 nmol/kg) or vehicle. Body weights wererecorded daily and used to administer the body weight-corrected doses ofpeptide throughout the study. Data are shown as mean±SEM in FIG. 7.

The control peptide (exendin-4) as well as Compound 8, significantlydecreased body weight gain at both doses (0.5 and 5 nmol/kg). Compound3, Compound 7, Compound 11 and Compound 12 significantly decreased bodyweight gain at the high dose (5 nmol/kg) but not at the low dose (0.5nmol/kg) (FIG. 7). Compound 6 significantly decreased body weight gainonly at the low dose (0.5 nmol/kg).

Example 9: Oral Glucose Tolerance 2, 4, 6, 8, 10 and 12 h after Dosingin High Fat Fed C57BL/6J

An oral glucose tolerance test (OGTT) was performed after subjecting theanimals to 4 hours of fasting. At 2, 4, 6, 8, 10 and 12 hours afterCompound 7 or vehicle dosing an initial blood sample were taken (t=−0min). Immediately thereafter, an oral dose of glucose (1 g/kg) wasgiven. BG levels were measured at t=15 min, t=30 min, t=60 min and t=90min. Immediately following blood sampling, all animals were sacrificedby CO₂ anesthesia followed by cervical dislocation. The study shows thatsubcutaneous administration with Compound 7 (10 nmol/kg) significantlyimproves glucose tolerance (measured as decreased AUC during an oralglucose tolerance test) 2, 4, 6, 8, 10 and 12 hours after dosing in highfat fed C57Bl/6J mice.

Example 10: Food Intake in Young Lean and Old Obese C57BL/6J Mice

C57BL/6J mice were on a high fat diet for 11 days and C57BL/6J mice wereon a high fat diet for 52 weeks.

On the day of the experiment, the mice were weighed and treated withs.c. injections of Exendin-4, Compound 7 or Vehicle at t=−0 h (8:00) andt=12 h (20:00). Immediately after treatment (t=0), pre-weighed food wereintroduced to the mice and the cumulative food intake was measured byweighing the remaining food after t=1, 2, 4, 8, 12 and 24 hours.

In the young lean mice, Compound 7 statistically significantly (p<0.05)reduced food intake during the 0-4, 0-8, 0-12 and 0-24 time periods.Exendin-4 statistically significantly (p<0.05) reduced food intakeduring the 0-2, 0-4, 0-8, 0-12 and 0-24 time periods.

In the old obese mice, Compound 7 statistically significantly (p<0.05)reduced food intake during the 0-2, 0-4, 0-8, 0-12 and 0-24 timeperiods. Exendin-4 statistically significantly (p<0.05) reduced foodintake in all time periods.

Example 11: Effect of 3 Weeks Subcutaneous Administration of GluGLP-1Agonist Compound 11 on Lipids in 30 Weeks High Fat Diet Feeded Mice

Effect of 3 weeks treatment of mice that have been on 30 weeks High FatDiet for 30 weeks prior treatment (s.c.) with vehicle (PBS), 10 nmol/kgexendin-4 or 10 nmol/kg Compound 11 twice daily for 3 weeks on lipids(FIG. 11). The effect was measured on LDL, HDL and triglycerids (CHO:Total Cholesterol; HDL: High Density Cholesterol; LDL: Low DensityCholesterol; TRIG: Triglycerides; HDL/LDL: Ratio between HDL and LDL).

Compound 11 significantly decreased cholesterol, HDL, LDL (P<0.001) andtriglycerides (P<0.05) significantly, while the ratio HDL/LDL wasincreased significantly (p<0.001) (FIG. 11). The HDL/LDL ratio isconsidered a risk indicator for heart disease. The the higher the ratio,the lower the risk of heart attack or other cardiovascular problems.

Example 12: Effect of Compound 11 on Hepatocyt cAMP Formation

All tested peptides behaved as full agonist with respect to GluRstimulated cAMP formation except of the pure GLP-1 agonists exendin-4and liraglutide. From the table it can observed that the rank order ofpotency is: Compound 1>glucagon>Compound 11>oxyntomodulin>>>exendin-4and liraglutide (Table 9).

Finally, no down regulation was observed of the E_(MAX) cAMP response atthe high concentrations, which is in contrast to what is observed in thehGluR HEK293 cells.

TABLE 9 Glucagon agonist effect on cAMP formation in human primarycultures. Compound GluR EC₅₀ (nM) Peptide No (1) (2) (log avg) Exendin-4∞ ∞ ∞ Glucagon 2.1 7.7 4.0 Oxyntomodulin 194.5 222.7 208.1 1 1.4 2.2 1.811 32.9 25.5 28.9 Liraglutide ∞ ∞ ∞

Example 13: Liver Weight of C57 Healthy Control Mice Treated for 2 Weeks

Repeated treatment with long-acting acylated dual GluGLP-1 agonists suchas Compound 11 do not give rise to change in liver size (enlarged orshrunken) compared with the non-acylated dual GluGLP-1 agonists compound1 (FIG. 12).

Example 14: HbA1c in db/db Mice after 28 Days

Diabetic (db/db) mice were treated with vehicle or compound 11 for 4weeks, and HbA1c was determined (Cobas® application note: A1C-2) inwhole blood samples (20 μl) collected from the treated mice. Results areshown in FIG. 13. The ΔHbA1c (%) was calculated for each mice bysubtracting its HbA1c (%) at start of treatment from HbA1c (%) at 4weeks. Treatment with compound 11 decreased ΔHbA1c (%) significantly.(P=0.03; Students t-test) compared to vehicle.

1. A compound having the formula:R¹—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formulaI (I) (SEQ ID NO: 4) His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-X27-X28-Ala-X30;

wherein X2 is selected from Aib and Ser; X12 is selected from Lys, Argor Leu; X16 is selected from Arg and X; X17 is selected from Arg and X;X20 is selected from Arg, His and X; X21 is selected from Asp and Glu;X24 is selected from Ala and X; X27 is selected from Leu and X; X28 isselected from Arg and X; X30 is X or is absent; wherein at least one ofX16, X17, X20, X24, X27, X28, and X30 is X; and wherein each residue Xis independently selected from the group consisting of Glu, Lys, Ser,Cys, Dbu, Dpr and Orn; wherein the side chain of at least one residue Xis conjugated to a lipophilic substituent having the formula: (i) Z¹,wherein Z¹ is a lipophilic moiety conjugated directly to the side chainof X; or (ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer,and Z¹ is conjugated to the side chain of X via Z²; with the provisothat Z is not HSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVEWLLRA (SEQID NO:5); or a pharmaceutically acceptable salt thereof. 2.-30.(canceled)
 31. A compound according to claim 1, wherein Z has theformula: (SEQ ID NO: 83) HSQGTFTSDYSKYLD-K(Hexadecanoyl-γ-Glu)-KAAHDFVEWLLRA; (SEQ ID NO: 84)HSQGTFTSDYSKYLDSKAAHDFVEWL-K(Hexadecanoyl-γ- Glu)-RA; (SEQ ID NO: 85)HSQGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)- DFVEWLLRA; (SEQ ID NO: 86)HSQGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl-γ- Glu)-A; (SEQ ID NO: 87)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- AAHDFVEWLLRA; (SEQ ID NO:88) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- AARDFVAWLLRA; (SEQ IDNO: 89) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- AAHDFVEWLLSA; (SEQID NO: 90) H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl- γ-Glu)-A;(SEQ ID NO: 91) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)-AAHDFVEWLLKA; (SEQ ID NO: 92)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- AAHDFVE()WLLK()A; (SEQ IDNO: 93) HSQGTFTSDYSKYLDS-K(Hexadecanoyl-γ-Glu)- AAHDFVEWLLRA; (SEQ IDNO: 94) H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-γ-Glu)- DFVAWLLRA; (SEQID NO: 95) H-Aib-QGTFTSDYSKYLDS-K(Dodecanoyl-γ-Glu)- AAHDFVEWLLSA; (SEQID NO: 96) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[3-aminopropanoyl])-AAHDFVEWLLSA; (SEQ ID NO: 97)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[8- aminooctanoyl])-AAHDFVEWLLSA;(SEQ ID NO: 98) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-ε-Lys)-AAHDFVEWLLSA; (SEQ ID NO: 99)HSQGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLLSA; (SEQ ID NO: 100)HSQGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)- AAHDFVEWLLSA; (SEQ ID NO: 101)HSQGTFTSDYSKYLDS-K([2-Butyloctanoyl]-γ-Glu)- AAHDFVEWLLSA; (SEQ ID NO:102) HSQGTFTSDYSKYLDS-K(Hexadecanoyl-[4- Aminobutanoyl])-AAHDFVEWLLSA;(SEQ ID NO: 103) HSQGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)- AAHDFVEWLLSA;(SEQ ID NO: 104) HSQGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLLSA; (SEQID NO: 105) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLLSA; (SEQ IDNO: 106) H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)- AAHDFVEWLLSA; (SEQID NO: 107) H-Aib-QGTFTSDYSKYLDS-K([2-Butyloctanoyl]-γ-Glu)-AAHDFVEWLLSA; (SEQ ID NO: 108)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[4- Aminobutanoyl])-AAHDFVEWLLSA;(SEQ ID NO: 109) H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl-γ-Glu)-AAHDFVEWLLSA; or (SEQ ID NO: 110)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-E)- AAHDFVEWLLSA;

wherein residues marked “( )” participate in an intramolecular bond; ora pharmaceutically acceptable salt thereof.
 32. A compound according toclaim 1, wherein Z has the formula: (SEQ ID NO: 111)H-Aib-QGTFTSDYS-K(Hexadecanoyl-isoGlu)- YLDSKAAHDFVEWLLSA; (SEQ ID NO:112) H-Aib-QGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)- KAAHDFVEWLLSA; (SEQ IDNO: 113) H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-isoGlu)- DFVEWLLSA; (SEQID NO: 114) H-Aib-QGTFTSDYSKYLDSKAAHDFV-K(Hexadecanoyl- isoGlu)-WLLSA;(SEQ ID NO: 115) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoLys)-AARDFVAWLLRA; (SEQ ID NO: 116)H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAKDFVEWLLSA; (SEQ ID NO:117) H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)- AAHDFVEWLLSA; (SEQ IDNO: 118) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAHEFVEWLLSA; (SEQID NO: 119) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)- AAEDFVEWLLSA;(SEQ ID NO: 120) H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLEA;

or a pharmaceutically acceptable salt thereof.
 33. A compound having theformula:R¹—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH²; and Z is a peptide having the formulaV (V) (SEQ ID NO: 12) His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-XIT-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-X28;

wherein X17 is X X28 is Ser or absent; wherein X is selected from thegroup consisting of Glu, Lys, and Cys; and wherein the side chain of Xis conjugated to a lipophilic substituent having the formula: (i) Z¹,wherein Z¹ is a lipophilic moiety conjugated directly to the side chainof X; or (ii) Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer,and Z¹ is conjugated to the side chain of X via Z².
 34. A compoundaccording to claim 33 wherein Z has the formula:H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLS  (SEQ IDNO:121);H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLL  (SEQ ID NO:122);or a pharmaceutically acceptable salt thereof.
 35. A compound having theformula:R¹—Z—R² wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formulaVI (VI) (SEQ ID NO: 131)His-Aib-Glu-Giy-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-XIT-Ala-Ala-His-Asp-Phe-Val-Glu- Trp-Leu-Leu-Ser-Ala;

wherein X17 is X; wherein X is selected from the group consisting ofGlu, Lys, and Cys; and wherein the side chain of X is conjugated to alipophilic substituent having the formula: (i) Z¹, wherein Z¹ is alipophilic moiety conjugated directly to the side chain of X; or (ii)Z¹Z², wherein Z¹ is a lipophilic moiety, Z² is a spacer, and Z¹ isconjugated to the side chain of X via Z²; or a pharmaceuticallyacceptable salt thereof.
 36. A compound according to claim 35 wherein Zhas the formula:H-Aib-EGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA  (SEQ ID NO:123); or a pharmaceutically acceptable salt thereof.
 37. A compositioncomprising a compound claim 1, or a pharmaceutically acceptable saltthereof, in a mixture with a carrier. 38.-39. (canceled)
 40. A method ofdiminishing weight gain or promoting weight loss in a subject, byadministering a compound of claim 1 to said subject, in an amountsufficient to diminish weight gain or promote weight loss.
 41. A methodof improving circulating glucose levels, glucose tolerance and/orcirculating cholesterol levels, lowering circulating LDL levels, and/orincreasing HDL/LDL ratio in a subject, by administering a compound ofclaim 1 to said subject, in an amount sufficient to improve circulatingglucose levels, glucose tolerance and/or circulating cholesterol levels,lowering circulating LDL levels, and/or increasing HDL/LDL ratio.
 42. Amethod of treatment of a condition caused or characterized by excessbody weight, and the treatment of obesity, morbid obesity, obesitylinked inflammation, obesity linked gallbladder disease, obesity inducedsleep apnea, metabolic syndrome, pre-diabetes, insulin resistance,glucose intolerance, type 2 diabetes, type I diabetes, hypertension,atherogenic dyslipidemia, atherosclerosis, arteriosclerosis, coronaryheart disease, peripheral artery disease, stroke or microvasculardisease in a subject, by administering a compound of claim 1 to saidsubject, in an amount sufficient to treat a condition caused orcharacterized by excess body weight, and to treat obesity, morbidobesity, obesity linked inflammation, obesity linked gallbladderdisease, obesity induced sleep apnea, metabolic syndrome, pre-diabetes,insulin resistance, glucose intolerance, type 2 diabetes, type Idiabetes, hypertension, atherogenic dyslipidemia, atherosclerosis,arteriosclerosis, coronary heart disease, peripheral artery disease,stroke or microvascular disease. 43-45. (canceled)
 46. The method ofclaim 40, wherein the compound is administered as part of a combinationtherapy with an agent for treatment of diabetes, obesity, dyslipidemia,or hypertension.
 47. The method of claim 46 wherein: a.) the agent fortreatment of diabetes is metformin, a sulfonylurea, a glinide, a DPP-IVinhibitor, a glitazone, insulin or an insulin analogue; b.) the agentfor treatment of obesity is a glucagon-like peptide receptor 1 agonist,peptide YY or analogue thereof, cannabinoid receptor 1 antagonist,lipase inhibitor, melanocortin receptor 4 agonist, or melaninconcentrating hormone receptor 1 antagonist; c.) the agent for treatmentof hypertension is an angiotensin-converting enzyme inhibitor,angiotensin II receptor blocker, diuretic, beta-blocker, or calciumchannel blocker and; d.) the agent for treatment of dyslipidemia is astatin, a fibrate, a niacin, and/or a cholesterol absorption inhibitor.48-50. (canceled)